Bacterial mutant BX065 and a method thereof

ABSTRACT

The present invention relates to a mutant bacteria  Xanthomonas oryzae  pathovar oryzae BXO65 with mutation at a site coding for enzyme shikimate dehydrogenase and a method of producing said mutant and also a method of screening inhibitors of bacterial pathogens with shikimate dehydrogenase pathway to develop bactericides.

FIELD OF THE INVENTION

[0001] The present invention relates to a mutant bacteria Xanthomonasoryzae pathovar oryzae BXO65 with mutation at a site coding for enzymeshikimate dehydrogenase. Also present invention relates to a method ofproducing said mutant. Further, a method of screening inhibitors ofbacterial pathogens with shikimate dehydrogenase pathway to developbactericides.

BACKGROUND AND PRIOR ART REFERENCES

[0002] Every year, plant pathogens cause billions of dollars worth ofdamage across the globe. Amongst the most destructive classes of plantpathogens are bacteria that grow within the xylem vessels of the hostplants. As opposed to fungal pathogens, effective bactericides are notavailable for protecting plants against bacterial pathogens.

[0003] Most of the available compounds are antibiotics (likestreptomycin and tetracycline) that are either not effective under fieldconditions or also affect the host plant. Moreover there is the concernthat the use of these antibiotics will result in the spread ofantibiotic resistance determinants amongst pathogens that cause diseasein humans and other animals. For e.g., tetracycline is one of the drugsof choice for use against Yersinia pestis (the plague bacterium). Theuse of tetracycline against plant pathogens might result in exposure ofYersinia pestis to this antibiotic as this bacterium is harbored by ratsthat populate agricultural fields. There is thus a need for theidentification of a new set of targets to develop novel bactericidesagainst plant pathogenic bacteria.

[0004]Xanthomonas oryzae pathovar oryzae (Xoo) is a bacterial pathogenthat causes bacterial leaf blight (BLB), a very serious disease of rice.Neither effective bactericides nor resistant rice cultivars areavailable to reduce yield losses due to this pathogen.

[0005]Xanthomonas oryzae pv. Oryzae causes bacterial leaf blight, aserious disease of rice. Worldwide at least 350 different plant diseasesare known to be caused by various xanthomonads (17). A characteristicfeature of the genus Xanthomonas is the production of yellow,membrane-bound pigments called xanthomonadins (28). The xanthomonadinsare initially thought to be carotenoids, but later they arecharacterized as a unique group of halogenated aryl polyene pigments (2,3).

[0006] The functional role of xanthomonadins is poorly understood. Thevast majority of pigment-deficient mutants that have been isolated fromseveral xanthomonads are prototrophs (20, 29) and virulence proficientupon wound inoculation (9, 20, and 29). Pigment-deficient mutants ofXanthomonas juglandis and X. oryzae pv. Oryzae have been reported to bemore sensitive to photobiological damage than the wold-type strains are(13, 22), suggesting that the pigment may provide protection againstphoto-damage.

[0007] An 18.6-kb region containing seven transcriptional units requiredfor xanthomonadin biosynthesis has been isolated from Xanthomonascampestris pv. Campestris (20, 21). One of the transcriptional units,pigB, encodes a diffusible factor that is involved in both pigment andextracellular polysaccharide production (21). The pigB mutants have alsobeen shown to be impaired for epipyhytic survival and host infection(22).

OBJECT OF THE PRESENT INVENTION

[0008] The main object of the present invention is to develop bacteriaXanthomonas oryzae pathovar oryzae BXO65 with mutation at a gene codingfor shikimate dehydrogenase.

[0009] Another main object of the present invention is to develop amethod of producing mutant bacterial strain Xanthomonas oryzae pathovaroryzae BXO65.

[0010] Yet another object of the present invention is to develop ascreening method to identify compounds inhibiting shikimatedehydrogenase pathway.

[0011] Still another object of the present invention is to identifybactericides against microbes having shikimate dehydrogenase pathway.

[0012] Still another object of the present invention is to developbactericides against bacteria Xanthomonas oryzae pathovar oryzae causingbacterial leaf blight (BLB) in rice.

SUMMARY OF THE PRESENT INVENTION

[0013] The present invention relates to a mutant bacteria Xanthomonasoryzae pathovar oryzae BXO65 with mutation at a site coding for enzymeshikimate dehydrogenase and a method of producing said mutant and also amethod of screening inhibitors of bacterial pathogens with shikimatedehydrogenase pathway to develop bactericides.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0014] Accordingly, the present invention relates to a mutant bacteriaXanthomonas oryzae pathovar oryzae BXO65 with mutation at a site codingfor enzyme shikimate dehydrogenase and a method of producing said mutantand also a method of screening inhibitors of bacterial pathogens withshikimate dehydrogenase pathway to develop bactericides.

[0015] In an embodiment of the present invention, a mutant bacteriaXanthomonas orzae pv. orzae (BXO65).

[0016] In another embodiment of the present invention, wherein saidmutant shows mutation in aroE gene coding for shikimate dehydrogenase.

[0017] In yet another embodiment of the present invention, wherein saidmutant is avirulent.

[0018] In yet another embodiment of the present invention, whereindoubling time of said mutant is about 25.7 hours.

[0019] In still another embodiment of the present invention, whereinsaid mutant produces very little Xanthomonadin pigments as compared towild type.

[0020] In still another embodiment of the present invention, whereinsaid mutant does not produce aromatic amino acids.

[0021] In further embodiment of the present invention, a method ofproducing a mutant bacterial strain Xanthomonas orzae pv. orzae (BXO65).

[0022] In still another embodiment of the present invention, whereinmutating BXO1 to produce BXO62.

[0023] In still another embodiment of the present invention, whereindeveloping rifampin resistance in said BXO62 to produce BXO65.

[0024] In still another embodiment of the present invention, whereinBXO1 is wild type bacterial strain of Xanthomonas orzae pv. Orzae.

[0025] In still another embodiment of the present invention, whereinBXO62 is pigment deficient bacterial strain of Xanthomonas orzae pv.Orzae.

[0026] In still another embodiment of the present invention, whereinsaid bacterial strains are grown at 28° C.

[0027] In still another embodiment of the present invention, whereinsaid bacterial strains are grown in medium selected from a groupcomprising peptone sucrose (PS) medium, and modified Miller's minimalmedium M4.

[0028] In still another embodiment of the present invention, whereinmutation is developed by using ethyl methanesulfonate.

[0029] In still another embodiment of the present invention, whereinrifampin is used at concentration ranging between 25 to 100 μg/ml.

[0030] In still another embodiment of the present invention, whereinBXO62 strain is obtained at a frequency of 1% of the surviving mutatedcells.

[0031] In further embodiment of the present invention, wherein saidmethod helps develop inhibitors against plant pathogenic bacteriaresponsible for Bacterial Leaf Blight (BLB) in rice.

[0032] In further embodiment of the present invention, a screening assaysystem to identify compounds to inhibit Shikimate dehydrogenase pathwayin bacteria to develop bactericide.

[0033] In still another embodiment of the present invention, whereinmixing shikimic acid and NADP as substrates.

[0034] In still another embodiment of the present invention, whereinadding test compounds to the said mixture.

[0035] In still another embodiment of the present invention, whereinmeasuring change in absorbance at 340 nm.

[0036] In still another embodiment of the present invention, whereinidentifying said inhibitors from the test compounds showing decrease inabsorbance.

[0037] In still another embodiment of the present invention, whereinabsorbance is measured spectrophotometrically.

[0038] In still another embodiment of the present invention, whereinmixing shikimic acid and NADP shows increase in absorbance.

[0039] In still another embodiment of the present invention, whereintest compounds with inhibitory action shows decrease in absorbance.

[0040] In still another embodiment of the present invention, whereinsaid method is used to develop inhibitors against pathogenic bacteriahaving shikimate dehydrogenase pathway.

[0041] In still another embodiment of the present invention, whereintest compounds are selected from a group comprising extracts, compounds,biomolecules, and pharmaceutical products.

[0042] In further embodiment of the present invention, whereindeveloping aroe gene mutant bacterial plant pathogen Xanthomonas oryzaepathovar oryzae named BXO65, with mutation in shikimate dehydrogenaseand thereby loss of virulence on rice crops.

[0043] In an embodiment of the present invention, applicants havedetermined that Xoo mutants that are defective in the production of theshikimate dehydrogenase enzyme are unable to cause disease on rice.

[0044] In another embodiment of the present invention, Shikimatedehydrogenase is a key enzyme in the aromatic amino acid biosyntheticpathway and the virulence deficiency appears to be due to the limitedavailability of aromatic amino acids within the xylem vessels of therice plant.

[0045] In yet another embodiment of the present invention, shikimatedehydrogenase can be used as a target to develop novel bactericidesagainst Xoo, and possibly against other bacterial pathogens. Thesebactericides can be in the nature of compounds that affect the shikimatedehydrogenase enzyme of the pathogen without affecting the host.

[0046] In still another embodiment of the present invention, theshikimate pathway is a good target for these purposes as it is absent inhumans and most animals. Therefore, compounds that target shikimatedehydrogenase may have minimal toxic affects with regard to exposure ofhumans and other animals to these compounds.

[0047] In still another embodiment of the present invention, thejudicious application of such compounds could help control yield lossesdue to Xoo as well as other bacterial pathogens that dwell in the xylemvessels of host plants. Since the shikimate pathway is absent in humans,an additional possibility is that inhibitors of shikimate dehydrogenasemay also be used against bacterial pathogens of humans.

[0048] In still another embodiment of the present invention,Xanthomonadins are yellow, membrane-bound pigments produced by membersof the genus Xanthomonas. Applicants identified an ethylmethanesulfonate-induced Xanthomonas oryzae pv. oryzae mutant (BX065)that is deficient for xanthomonadin production and virulence on rice, aswell as auxotrophic for aromatic amino acids (Pig⁻Vir⁻Aro⁻). Reversionanalysis indicated that these multiple phenotypes are due to singlemutation.

[0049] In still another embodiment of the present invention, a genomiclibrary of the wild-type strain is used to isolate a 7.0-kb clone thatcomplements BX065. By transposon mutagenesis, marker exchange, sequenceanalysis, and subcloniong, the complementing activity is localized to a849-bp open reading frame (ORF). This ORF is homologous to the aroEgene, which encodes shikimate dehydrogenase in various bacterialspecies.

[0050] In still another embodiment of the present invention, Shikimatedehydrogenase activity is present in the wild-type strain and the mutantwith the complementing clone, whereas no activity is found in BX065.This clone also complemented an Escherichia Coli aroE mutant forporototrophy, indicating that aroE is functionally conserved in X.oryzae pv, oryzae and E. Coli.

[0051] In still another embodiment of the present invention, thenucleotide sequence of the 2.9-kb region containing aroE revealed that aputative DNA helicase gene is located adjacent to aroE. Applicantsresults indicate that aroE is required for normal levels of virulenceand xanthomonadin production in X. oryzae pv. Oryzae.

LIST OF THE ACCOMPANYING DRAWINGS

[0052]FIG. 1 represents structure of Xanthomonadin I and spectra ofcrude pigment extracts.

[0053]FIG. 2 represents Virulence of X.oryzae. pv oryaze strains forrice.

[0054]FIG. 3 represents nucleotide sequence of X. oryzae. pv oryazearo.E gene and deduced amino acid sequence.

[0055] In still another embodiment of the present invention, theapplicants isolated an ethyl methanesulfonate (EMS)-inducedpigment-deficient mutant of X. oryzae pv. Oryzae that is also virulencedeficient and auxotrophic for aromatic amino acids. A genomic clone thatrestores pigmentation, prototrophy, and virulence to this mutant isisolated by functional complementation.

[0056] In still another embodiment of the present invention,characterization of this clone revealed that shikimate dehydrogenase, anenzyme in the aromatic amino acid biosynthetic pathway, is required fornormal levels of pigment production and virulence in X. oryzae pv.Oryzae.

[0057] In still another embodiment of the present invention, allbacterial strains and plasmids used are listed in Table 1. TABLE 1Strains and Plasmids used in this study Strain or plasmid RelevantCharacteristics^(a) Reference or source E. coli strains DH5α F′ endA1hsdR17 (r⁻m⁺) supE44 thi-1 recA1 gyrA Lab collection relA1 f80dlacZDM15(lacZYA-argF) U169 S17-1 RP4-2Tc::Mu-Kn::Tn7 pro hsdR recA 26 AB2835AeroE353 30 Plasmids Pbluescript (KS) Ap^(r) Stratagene, La Jolla,Calif. PHM1 Sp^(r) Sm^(r) cos parA IncW derivative of pRI40 11 PUFR034IncW Nm^(r) Tra⁻Mob⁺ mob(P) LacZα⁺ Par⁺ cos 8 PAG4 pUFR034 + 7.0-kbEcoRI fragment encoding aroE This study PAG5 pBluescript (KS) = 7.0-kbEcoRI fragment encoding This study aroE PAG7 Pufr034 + 1.2-kb EcoRIfragment encoding aroE This study PAG8 pAG4-aroE1::Tn3-HoHo1 This studyPAG9 pAG5-aroE2::mTn7 This study PAG10 pAG5-aroE3::mTn7 This study PAG11pHM1+ aroE2::mTn7 (in the 7.0-kb EcoRI fragment) This study PAG12 pHM1+aroE3::mTn7 (in the 7.0-kb EcoRI fragment) This study X. oryzae pv.Oryzae strains BXO1 Laboratory wild type, Indian isolate Lab collectionBXO43 rif-2, derivative of BXO1 Lab collection BXO62 pig-10, obtained byEMS mutagenesis of BXO1 This study BXO65 rif-5, derivative of BXO62 Thisstudy BXO1704 BXO65/pAG4 This study BXO1706 aroE1::Tn3-HoHo1 rif-2 Thisstudy BXO1707 aroE2::mTn7 rif-2 This study

[0058] In still another embodiment of the present invention, X. oryzaepv. Oryzae strains are grown at 28° C. in either peptone sucrose (PS)medium or modified Miller's minimal medium M4. Escherichia coli strainsare grown in Luria-Bertani medium at 37° C.

[0059] In still another embodiment of the present invention, thefollowing concentrations of antibiotics are used: rifampin, 50 μg ml−¹;spectinomycin, 50 μg ml−¹; ampicillin, 100 μg ml−¹; kanamycin 50 μgml−¹; chloramphenicol 20 μg ml−¹, streptomycin,50 μg ml−¹ andcycloheximide, 75 μg ml−¹.

[0060] In still another embodiment of the present invention, EMS (SigmaChemical Co., St. Louis, Mo.) mutagenesis of X. oryzae pv. Oryzae isdone as described for E. coli by Miller.

[0061] In still another embodiment of the present invention, twentymicroliters of a cell suspension from a mutagenized and washed X. oryzaepv. Oryzae cell preparation is inoculated into 2 ml of PS medium andincubated at 28° C. for 24 h before the cells are dilution plated toobtain single colonies of PSagar (PSA) plates.

[0062] In still another embodiment of the present invention,pigment-deficient mutants are obtained at a frequency of 1% of thesurviving cells. One pigment-deficient mutant failed to grow on minimalmedium and the auxotrophic phenotype is diagnosed as a deficiency of allthree aromatic amino acids by using the pool plate method.

[0063] In still another embodiment of the present invention, spontaneousprototrophic revertants and rifampin-resistant (Rif′) derivatives of X.oryzae pv. Oryzae strains are obtained by plating saturated cultures(approximately 10⁸ CFU/plate) on plates containing minimal medium andplates containing PSA plus rifampin, respectively.

[0064] In still another embodiment of the present invention, apigment-deficient mutant (BXY062) that is auxotrophic for all threearomatic amino acids is isolated after EMS mutagenesis of the BXO1strain.

[0065] In still another embodiment of the present invention, to aid inthe subsequent analysis, spontaneous Rif^(r) derivatives of BXO1 andBX062 designated BXO43 and BXO65 respectively, are obtained.

[0066] In still another embodiment of the present invention, the pigmentproduction, prototrophic-auxotrophic, and virulence properties of theRif^(r) derivatives are similar to those of their respective parentstrains (data not shown). Pigment is extracted from strains BX043 andBX065 and quantified. BX065 produces approximately 23% of the pigmentproduced by BX043 (Table 2). TABLE 2 Pigment production and shikimatedehydrogenase activity in various X. oryzae pv. Oryzae strainsStrain^(a) Shikimate dehydrogenase Pigment production activityprotein)^(c) (optical density at 445 nm)^(b) (mU/mg of BX043  0.632 ±0.006^(d) 5.35 ± 0.57 BX 065  0.15 ± 0.018 NDA^(e) BX 01704 0.816 ±0.03  65.3 ± 6.07

[0067] In still another embodiment of the present invention, anabsorption spectrum of the pigment extracted from the BX043 strainshowed a characteristic structure with peak at 445 nm and shoulders at425 and 470 nm (FIG. 1). These features are missing from the absorptionspectrum of the BX065 strain. (FIG. 1.)

[0068] In still another embodiment of the present invention, thevirulence characteristics of these strains were assayed with rice leavesas described in Materials and Methods.

[0069] Lengths of lesions caused by BXO43 and BXO65 are measured 10 and15 days after inoculation.

[0070] In still another embodiment of the present invention, it isapparent that BXO65 is severely virulence deficient (Vir−). Prototrophicrevertants of BXO65 are isolated and are found to have regained pigmentand virulence proficiency (data not shown). Simultaneous reversionsuggests that a single mutation is responsible for the pleiotropicphenotype of BXO65.

[0071] In still another embodiment of the present invention,forty-day-old greenhouse-grown rice plants of susceptible rice cultivarTaichung Native-1 are inoculated by clipping leaf tips with sterilescissors dipped in saturated cultures (10⁸ cells/ml) of X.oryzae pv.Oryzae.

[0072] In still another embodiment of the present invention, when thismethod is used, approximately 10⁸ cells are deposited at the site ofinoculation. The plants are incubated in a greenhouse with minimum andmaximum temperatures of approximately 25 and 30° C., respectively, and arelative humidity of approximately 60%.

[0073] In still another embodiment of the present invention, lesionlengths are measured at regular intervals. No lesions are observed incontrol experiments in which the leaves are inoculated with scissorsdipped in water.

[0074] In still another embodiment of the present invention, theprocedure described previous for extraction of xanthomonadin from X.juglandis is used, with some modifications, X. oryzae pv.

[0075] In still another embodiment of the present invention, Oryzaecultures are grown to the stationary phase and xanthomonadin isextracted in chloroform-methanol (2:1) by shaking for 3 h at roomtemperature. The amount of pigment produced per 100 mg. (dry weight) ofbacterial cells is expressed as the absorbance (optical density at 445nm) of the crude pigment extracts.

[0076] In still another embodiment of the present invention, applicantsalso quantified the pigment by assuming that the structure and molarextinction coefficient of xanthomonadin from X. oryzae pv. Oryzae areidentical to those of a xanthomonadin from X. juglandis. Similarconclusions could be drawn by using either method for pigmentestimation.

[0077] In still another embodiment of the present invention, a partialEcoRI-digested genomic library of applicant's laboratory wild-typeX.oryze pv. Oryze strain, having an average insert size of 30 kb, isprepared in the broad-host range Km^(r) cosmid cloning vector pUFR034.

[0078] In still another embodiment of the present invention, a total of960 clones from this library are transferred from E. coli DH5α to S17-1by using pRK600 as a helper. Genomic clones from the library in S17-1are mobilized in pools of 12 clones each into the pigment-deficientmutant by performing biparental mating.

[0079] In still another embodiment of the present invention,transconjugants that appeared on PSA-rifampin-kanamycin selection platesare replica plated onto minimal medium plates containing kanamycin toidentify prototrophic and pigment-proficient colonies.

[0080] In still another embodiment of the present invention, clones froma cosmid genomic library of BXO1 are mobilized into BX065 in 55 poolscontaining 12 clones each.

[0081] In still another embodiment of the present invention, weidentified three donor pools which yielded Aro⁺ Pig⁺ exconjugants.Individual clones from the three pools are mobilized into BX065, whichled to identification of three independent complementing clones, pAG1,pAG2, and pAG3.

[0082] In still another embodiment of the present invention, the EcoRIrestriction patterns of the three plasmids revealed that there are threecommon Eco RI fragments (7.0, 4.0, and 1.5 kb). When the 7.0-kb fragmentis subcloned (pAG4) and mobilized into BX065, it is found to contain thecomplementing activity.

[0083] In still another embodiment of the present invention, theabsorption spectrum of the pigment extracted from BX01704 (BX065/pAG4)is identical to that of BX043 (FIG. 1), and the amount of pigmentproduced is estimated to be 30% more than the amount produced in thewild type (Table 2).

[0084] In still another embodiment of the present invention, this couldhave been due to the presence of the aroE gene on a multicopy plasmid inBXO1704. The pAG4 plasmid also complemented BX065 partially (˜60%) forvirulence on rice (FIG. 2). This partial complementation could have beena result of instability of the clone because of the absence ofantibiotic selection inside the plant.

[0085] In still another embodiment of the present invention, transposon3 insertions are obtained with the pAG4 clone by using Tn3-HoHo1, withsome modifications. Plasmids are isolated, and the insertions arelocalized on either the insert or the vector DNA by restrictionanalysis.

[0086] In still another embodiment of the present invention, the 7.0-kbEcoRI fragment in pAG4 is also cloned into the EcoRI site ofpBluescript(KS) to obtain pAG4 and is mutagenized by mini-transposon 7(mTn7) derivatives by using an in vitro transposition kit (GenomePriming System; New England Biolabs [NEB], Beverly, Mass.).

[0087] In still another embodiment of the present invention, the mTn7element encodes kanamycin resistance. Two different mTn7 insertions onthe cloned DNA in plasmids pAG9 and pAG10 (Table 1) are found to be inthe aroE gene (see below). These two insertions are cloned into theshuttle vector pHM1(Sp^(r)) (Table 1) by usisng EcoRI restriction sites(EcoRI does not cut within the mTn7 element) to obtain plasmids pAG11and pAG12.

[0088] In still another embodiment of the present invention, PlasmidspAG11 and pAG12 and the derivatives of Pag containing Tn3-HoHo1insertions are mobilized individually into BX0-43 for marker exchange.

[0089] In still another embodiment of the present invention, markerexchange is done by growing the cells in either PS-ampicillin medium(for Tn3-HoHo1) or PS-kanamycin medium for (for mTn7) for more than 30generations by serial passage.

[0090] In still another embodiment of the present invention, coloniesthat are either Ap^(r) Km^(s) (for Tn3-HoHo1) or Km^(r) Sp^(s) (formTn7) are analyzed by Southern hybridization to confirm that markerexchange had occurred as expected.

[0091] In still another embodiment of the present invention, the 7.0-kbEco RI fragment in the pAG4 clone is subjected to mutagenesis by usingTn3-HoHo1 and mTn7.

[0092] In still another embodiment of the present invention, sevenindependent Tn3-HoHo1 insertions are obtained on pAG4, and one of them,aroE1::Tn3-HoHo1, affected the ability to complement BX065 (data notshown). This insertion is marker exchanged in the BX043 background. Themarker exchange mutant BXO1706 is found to be Pig⁻(FIG. 1), Vir⁻(datanot shown), and auxotrophic for aromatic amino acids.

[0093] In still another embodiment of the present invention,introductions of pAG4 into BXO1706 restored the Pig⁺ Vir⁺ Aro⁺ phenotype(data not shown). This indicated that aroE1::Tn3-HoHo1 disrupted atranscriptional unit that is required for pigmentation, virulence, andprototrophy.

[0094] In still another embodiment of the present invention, Markerexchange mutants obtained with six other Tn3-HoHo1 insertions are Pig⁺Vir⁺ Aro⁺. Twenty-nine mTn7 insertions are obtained for the 7.0-kb DNAcloned in pAG5 (see Materials and Methods). Two of these insertions,aroE2::mTn7 AND aroE3::mTn7 in plasmids pAG9 and pAG10, respectively,are found to be in the aroE open reading frame (ORF). These twoinsertions along with flanking 7.0-kb sequences are cloned in the EcoRIsite of the plasmid shuttle vector pHM1 to give plasmids pAGI 1 andpAG12.

[0095] In still another embodiment of the present invention, both pAG11and pAG12 failed to complement BX065, indicating that aroE2::mTn7 andaroE3::mTn7 abolished the complementing ability of the 7.0 kb genomicfragment. When the aroE2::mTn7 insertion is marker exchanged in theBX043 background, it produced the Pig⁻ Vir⁻ Aro⁻ mutant strain BXO1707(data not shown). The aroE3::mTn7 insertion could not be markerexchanged, most likely because of limited homology on one side of theinsertion (FIG. 3).

[0096] In still another embodiment of the present invention, plasmid DNAis isolated by the alkaline lysis method.

[0097] In still another embodiment of the present invention, Restrictiondigestions are performed by using enzymes obtained from NEB asrecommended by the supplier. Primers that are outwardly directed fromTn3-HoHo1 are used to obtain the sequence of DNA flanking the Tn3-HoHo1insertions. mTn7-specific primers provided in the Genome Priming Systemkit from NEB are used to sequence the mTn7 insertion sites. M13 forwardand reverse primers are used to sequence to ends of a 7.0-kb insert inclone pAG5.

[0098] In still another embodiment of the present invention, thesequencing reactions, electrophoresis, and sequence data analyses areperformed with an ABI Prism 377 automated DNA sequencer (Perkin-Elmer,Foster City, Calif.).

[0099] In still another embodiment of the present invention, a homologysearch in the database is performed through the National Center forBiotechnology Information by using the BLAST algorithm (1). Promoterprediction is performed by using software at the Baylor College ofMedicine search launcher (www.hisc.bcm.tmc.edu).

[0100] In still another embodiment of the present invention, thesequence of a 2.953-kb region containing aroE is determined. Thissequence has been submitted to GenBank with accession no. AF258797.

[0101] In still another embodiment of the present invention, a 2.953-kbregion cloned in pAG5 is sequenced by using transposon-specific primers,as well as primer walking.

[0102] In still another embodiment of the present invention, theinsertions aroE1::Tn3-HoHo1, aroE2::mTn7, and aroE3::mTn7 are found tobe present in a 849-bp ORF with the potential to encode a 283-amino-acidprotein (FIG. 3).

[0103] In still another embodiment of the present invention, a homologysearch using the BLAST algorithm revealed that the ORF is homologous toaroE, which encodes shikimate dehydrogenase, an enzyme in the aromaticamino acid biosynthetic pathway of various bacterial species.

[0104] In still another embodiment of the present invention, maximumhomologies are found with aroE from Pseudomonas aeruginosa(EMBL/GenBank/DDBJ database accession no. X85015), Neisseriameningitidis, E. coli, and Haemophilus influenzae, as shown in Table 3.TABLE 3 Homology of the X. oryzae pv. Oryzae aroE gene with the aroEgenes of other bacterial species Length of gene product (amino %similarity Organism acids) (% identity) E value Accession no P.aeruginosa 274 58(45) e-54 X85015 N. meningitidis 269 57(41) e-50 U82840E. Coli 272 58(44) e-50 U18997 H. influenzae 272 53(38) e-35 U32748

[0105] In still another embodiment of the present invention, promoterprediction, as described in Materials and Methods, indicated thepresence of −35 and −10 promoter regions 100 and 75 nucleotides upstreamof the putative ATG start codon, respectively.

[0106] In still another embodiment of the present invention, within theORF the aroE1::mTn3-HoHo1, aroE2::mTn7, and aroE3::mTn7 insertions arelocated at the 634^(th), 350^(th), and 15^(th) nucleotides, respectively(FIG. 3). A PstI restriction site is identified 200 bp downstream of thearoE stop condon. This site is used to clone a 1.2-kb EcoRI-PstIfragment that included the aroE ORF to obtain plasmid pAG7. When pAG7 isintroduced into BX065, this minimal fragment is sufficient to confer thePig⁺ Vir⁺ Aro⁺ phenotype (data not shown).

[0107] In still another embodiment of the present invention, in the2.953-kb sequence, another ORF downstream of aroE is identified by usingthe BLAST algorithm. This incomplete ORF started at 1,546^(th)nucleotide and showed very strong homology in the 1,407 bp sequenced(55% similarity and 36% identity at the amino acid level) to dinG (DNAdamage-inducible gene G), which enclodes an ATP-dependent helicase inE.coli.

[0108] In still another embodiment of the present invention, E. coliaroE mutant strain AB2835 (30) is transformed individually with pAG4 andpAG5 and with plasmids pAG8, pAG9, and pAG10 having transposoninsertions in the aeroE ORF (Table 1).

[0109] In still another embodiment of the present invention, PlasmidspAG4 and pAG5 complemented AB2835 for growth on minimal medium, whereasthe other plasmids having an insertion in the aroE ORF are not able todo so.

[0110] In still another embodiment of the present invention, all strainsgrew on minimal medium supplemented with the three aromatic amino acidsand the aromatic vitamins p-aminobenzoic acid and p-hydroxybenzoic acid.Plasmid curing from strain AB2835/pAG4 resulted in a loss of prototrophy(data not shown).

[0111] In still another embodiment of the present invention, celllysates are prepared as described by Chaudhuri and Coggins, and proteincontents are estimated by the Bradford method (5). Shikimatedehydrogenase activities in cell lysates of X.oryzae pv. Oryzae strainsare assayed as described previously for E.coli.

[0112] In still another embodiment of the present invention, Shikimicacid and NDAP are used as substrates, and reduction of NADP to NADPH isassayed by monitoring increase in absorbance at 340 nm. One unit ofenzyme activity is defined as 1 μmol of NADPH produced per min.

[0113] In still another embodiment of the present invention, proteinextracts are prepared from the wild-type and aroE mutant strains of X.oryzae pv. Oryzae, and shikimate dehydrogenase activity is measured (seeMaterials and Methods).

[0114] In still another embodiment of the present invention, theMichaelis constant of this enzyme for shikimic acid is calculated to be5×10⁻⁵M, a value very similar to that E. coli (32). The enzyme activityis found to be 5.4 mU/mg of protein in wild-type strain BX043 (Table 2).

[0115] In still another embodiment of the present invention, no activityis detected in aroE mutant-strain BX065 or in BX01706(aroE1::Tn3-HoHo1)or BXO1707 (aroE2::mTn7). The BXO1704 strain (BX065/pAG4) had anactivity of 65 mU/mg of protein, approximately 12 times that of BX043(Table 2).

[0116] In still another embodiment of the present invention,cross-feeding of BX065 with 15 independently isolated EMS-inducedprototrophic pigment-deficient mutants is tested by streaking the mutantstrains adjacent to BX065 in pairwise combinations on PSA plates.

[0117] In still another embodiment of the present invention, developmentof a yellow color in BXO65 at the adjacent colony boundary is consideredto indicate cross-feeding for pigmentation.

[0118] In still another embodiment of the present invention, BXO65 couldnot be cross-fed for pigmentation by wild-type strain BX043, whereas 8of 15 EMS-induced prototrophic pigment-deficient mutants could do so.

[0119] In still another embodiment of the present invention, none of the15 prototrophic pigment-deficient mutants could be cross-fed by eachother BX043, or BX065 for pigmentation.

[0120] In still another embodiment of the present invention, mutationsin the aroE gene of X. oryzae pv. Oryzae result in reduced levels ofxanthomonadin production and virulence. To the best of our knowledge,this is the first report of a mutation affecting a specific step in ageneral metabolic pathway that also affects xanthomonadin biosynthesis.

[0121] In still another embodiment of the present invention, insertionalinactivation of any of the seven transcriptional units previously shownto be required for xanthomonadin biosynthesis in x.campestris pv.Campestris has not been reported to cause any nutrient deficiences.

[0122] In still another embodiment of the present invention, also,clones containing the aroE gene of X. oryzae pv. Oryzae do not show anyhomology with the pig gene cluster of either X.campestris pv. Campestrisor X.oryzae pv. Oryzae. This indicates that the aroE gene is from adifferent genomic locus than the gene cluster that was previously shownto be required for xanthomonadin biosynthesis.

[0123] In still another embodiment of the present invention, shikimatedehydrogenase catalyzes the conversion of dehydroshikimate to shikimicacid. How might shikimate production be related to xanthomonadinbiosynthesis? One possibility, as previously suggested, is that thearomatic ring in xanthomonadin may be derived from the shikimatepathway. This would be interesting because the aromatic ring in aromaticcarotenold pigments is derived from cyclization of the polyene chain andnot from the shikimate pathway. However, we have observed that shikimicacid facilitates neither growth on minimal medium nor pigmentation inaroE mutants (Goel and sonti, unpublished results). This might bebecause aroE mutants of X. oryzae pv. Oryzae are deficient in shikimicacid uptake, as reported previously for E. coil aroE mutants.

[0124] In still another embodiment of the present invention, theresidual amount of pigment made in the aroE mutants may be derivedeither from a very small amount of shikimic acid produced by spontaneousconversion of dehydroshikimate to shikimic acid or through a secondminor pathway. It is also possible that the pigment produced by the aroEmutants is devoid of the aromatic ring.

[0125] In still another embodiment of the present invention, detailedstructural analysis and biochemical experiments, such as feedingwild-type cells with labelled shikimic acid and monitoring theincorporation of the label, are required to address these issues.

[0126] In still another embodiment of the present invention, severalEMS-induced pigment-deficient mutants are able to cross-feed aroE mutantstrains for pigment production. This suggests that a diffusible compoundthat can enter the pigment biosynthetic pathway accumulates in thesestains.

[0127] In still another embodiment of the present invention, asxanthomonadins are membrane-bound pigments, the compound or intermediatemust act prior to commitment to the membrane-bound state.

[0128] In still another embodiment of the present invention, theinability of the wild-type strain and some of the EMS-induced mutants tocross-feed aroE strains suggests that the compound does not accumulatein these strains.

[0129] In still another embodiment of the present invention, theinability of the prototrophic EMS-induced pigment-deficient mutants tocross-feed each other suggests that they are defective in reactions thatinvolve commitment to either the membrane-bound state or subsequentsteps in xanthomonadin production and transport.

[0130] In still another embodiment of the present invention, thevirulence deficiency of the aeroE mutants is most likely due to a growthdefect. The in planta doubling times for the wild type, aroE mutantBXO65, and a mutant with a complementing clone are 8.6, 25.7, and 10.5h, respectively (data not shown).

[0131] In still another embodiment of the present invention, this is thefirst report which suggests that one or more aromatic amino acids may belimiting for growth of X. oryzae pv. oryzae in rice plants. A virulencedeficiency has previously been reported to be associated with eitherarginine or leucine auxotrophy in X. oryzae pv. oryzae.

[0132] In still another embodiment of the present invention, the resultsalso suggest that shikimate dehydrogenase can be used as a target todevelop novel bactericides against X. oryzae pv. oryzae. Thesebactericides can be compounds that affect the shikimate dehydrogenase ofthe pathogen without affecting the host. The shikimate pathway may be agood target for this purpose as it is absent in mammals.

[0133] In still another embodiment of the present invention, it ispertinent to note that effective bactericides are not available for useagainst X. oryzae pv. oryzae and that judicious application of suchcompounds could help control yield losses due to this devastating ricepathogen.

[0134] The details of the present invention are illustrated below withthe help of examples but should not be construed to limit the scope ofthe invention.

EXAMPLES Example 1

[0135] Overexpression and Purification of Shikimate Dehydrogenase Enzymeof Xanthomonas oryzae pathovar oryzae.

[0136] The plasmids pAG7 containing aroE and the pGEX2T (PharmaciaBiotech, Upsaala, Sweden) are purified from cultures of Escherichia coliby the alkaline lysis method (25). PCR (Polymerase Chain Reaction)primers for the aroE gene are designed, by methods familiar to thosewell versed in the art, to incorporate the sites for BamHl and EcoRL.PCR is performed with Vent Polymerase (NEB) using pAG7 DNA as templateto obtain a single fragment of approximately 870 bp. This fragment isdigested with restriction enzymes BamH1 and EcoRI obtained from NEB.This fragment is cloned into the BamHI and EcoRI sites of pGEX2T usingstandard procedures as described (25) to generate a fusion of theshikimate dehydrogenase (aroE) alongwith the gene forglutathione-S-transferase that is encoded on pGEX2. Exponentiallygrowing E. coli cultures containing this recombinant aroE plasmid areinduced with IPTG (Isopropyl thio galactoside; a potent inducer of thepromoter to which aroE gene is fused) to produce large amounts of theshikimate dehydrogenase fusion protein. The uninduced plasmid does notproduce the fusion protein. The cells are lysed and the overexpressedshikimate dehydrogenase-glutathione S-transferase fusion protein ispurified using glutathione agarose beads as per manufacturer's(Pharmacia) instructions. Shikimate dehydrogenase protein is obtained bycleaving the fusion protein with thrombin as per manufacturer's(Pharmacia) instructions.

Example 2

[0137] Development of a Screening Procedure for Identifying Inhibitorsof Shikimate Dehydrogenase Enzyme of Xanthomonas oryzae Pathovar oryzae.

[0138] The purified shikimate dehydrogenase enzyme can be used in enzymeassays (32) to identify chemicals that are inhibitory to the activity ofthe enzyme. The assay involves use of Shikimic acid and NADP assubstrates. The reduction of NADP to NADPH is followed by monitoringincrease in absorbance at 340 nm. The inhibitors would be chemicals thatwould prevent/reduce enzyme mediated conversion of NADP to NADPH asmonitored by following absorbance at 340 nm. The total reaction volumewould be 100μ liters, sufficient to be accommodated in the 250μ literwells of ELISA plates. The absorbance can be followed on an ELISAspectrophotometer. The reaction is rapid and proceeds to saturation inthirty seconds to one mimjte. This facilitates the screening of a largenumber of candidate inhibitor molecules. The procedure can be automated,wherein liquid handling systems can be used for pipetting variousreaction ingredients including enzyme, substrates, buffer, inhibitors,etc. The procedure for spectrophotometric analysis can also beautomated, facilitating the screening of a large number of chemicallibraries. These procedures for automation are routinely used in drugdiscovery programmes all over the world and can be adapted to screen forshikimate dehydrogenase inhibitors using the above assay with thepurified enzyme.

Example 3

[0139] Development of a Screening Procedure for Identifying Inhibitorsof Shikimate Dehydrogenase Enzyme of Xanthomonas oryzae Pathovar oryzaeUsing the Cloned X. oryzae Pathovar oryzae aroE Gene (PAGS).

[0140] The principle of this method is that a strain carrying shikimatedehydrogenase gene on a high copy number plasmid (pAG5; pBluescript isthe vector) will produce higher amounts of shikimate dehydrogenase andcan therefore tolerate higher concentrations of shikimate dehydrogenaseirihibitor. The host strain would be an E. coli wild type straincontaining either pAG5 (table 1) or the pBluescript vector without addedinsert. The use of E. coli is doubly advantageous because it growsfaster than X. oryzae pv. oryzae and the high copy number plasmid (copynumber of 500-1000/cell) can replicate in E. coli and not in X. oryzaepv. oryzae. Results presented demonstrate that the X. oryzae pv. oryzaegene is expressed and will functionally complement an E. coli aroEmutant. The criteria for a shikimate dehydrogenase enzyme inhibitorwould be retardation of growth on minimal medium of an E. coli strainthat carries the high copy number plasmid (without aroE gene) witheither little or no effect on a strain that has the aroE gene on a highcopy plasmid. The growth assays can be performed in 96 well ELISA platesand growth can be followed by change in absorbance at 600 nm in ELISAspectrophotometer. Again, this is a procedure that can be automated.

Example 4

[0141] Assay for Inhibition of Growth of Xanthomonas oryzae PathovarOryzae by Inhibitors of Shikimate Dehydrogenase.

[0142] The inhibitors of shikimate dehydrogenase identified by assaysconducted as described in Examples 2 and 3 are screened for theirability to inhibit growth of Xanthomonas oryzae pathovar oryzaeonjninimal mediumprepared as described (15). Saturated cultures of thewild type X. oryzae pathovar oryzae grown in minimal medium are dilutedten fold in double distilled water and plated onto minimal medium agarplates containing different concentrations of the inhibitor in thepresence or absence of aromatic amino acids. An inhibitor of shikimatedehydrogenase should prevent growth of X. oryzae pathovar oryzae only onminimal medium that is not supplemented with aromatic amino acids. Thisinhibitor should also inhibit xanthomonadin production which can beassayed as lack (or reduced amounts) of the characteristic yellowpigmentation. The assay for reduction in xanthomonadin production isperformed on Peptone Sucrose Agar (29) medium.

Example 5

[0143] Assay for Effects on Rice Plants of Identified Inhibitors of X.oryzae Pathovar Oryzae Shikimate Dehydrogenase.

[0144] Forty to fifty day old green house grown rice plants of cultivarsTaichung Native-1 and BPT5204 (a fine quality rice from South India) aresprayed with solutions of different concentrations of the inhibitor andcompared with those that are sprayed only with the solvent (water). Theplants are subjected to a second round of spraying, 36 hours after theinitial spaying. The plants are examined at regular intervals, prior toand after the second round of spraying, for symptoms of chlorosis (lossof green coloration), stunting (growth inhibition) and at later stagesfor defects in the emergence of panicle (rice inflorescence). X. oryzaepathovar oryzae is also a seed borne pathogen and therefore bactericideswould also be used to used to rid the seeds of infestation by thispathogen. Therefore any potential bactericide would have to be examinedto determine if it has adverse effects on seed germination. For thispurpose, rice seeds would be germinated in growth chambers orincubators, at 28° Centigrade, on moistened filter paper containingdifferent concentrations of the inhibitor. Germination is assayed after72-96 hours as emergence of radicals (roots) and shoots. Shikimatedehydrogenase assays can also be performed as described hi Example 2, oncrude protein extracts of rice leaves, in order to determine if any ofthe tested compounds will affect rice shikimate dehydrogenase activity.

Example 6

[0145] Assay for Amelioration of Bacterial Leaf Blight Symptoms of Riceby Identified Inhibitors

[0146] Forty to fifty day old green house grown rice plants of cultivarsTaichung Native-1 and BPT5204 (a fine quality rice from South India) areinoculated with saturated cultures of X. oryzae pathovar oryzae byeither clipping (14) or dipping leaves in bacterial culture. In thelatter method of infection, which is more akin to natural conditions,the bacterium must enter the plant through natural openings calledhydathodes. Twelve hours after inoculation an optimum concentration ofthe inhibitor (identified in Examples 4 and 5) that would not inhibitgrowth of rice but would inhibit X. oryzae pathovar oryzae is sprayedover these plants. Control plants would also be sprayed with water alone(no inhibitor). Small amounts of a surfactant such as Tween 20 will alsobe added to test solutions as well as the control in order to facilitateabsorption by the leaf. This procedure is repeated 36 hours later. Aferan additional week on the greenhouse bench, the leaves are examined forbacterial leaf blight symptoms. The efficiency of infection (number ofleaves exhibiting symptoms of bacterial leaf blight/total number ofinfected leaves) as well as the length of the lesions are estimated. Aneffective inhibitor would be one that reduces efficiency of infection aswell as lesion length.

Example 7

[0147] A Procedure for Identification of Inhibitors that a/feetShikimate Dehydrogenase of X. oryzae Pathovar Oryzae but not ShikimateDehydrogenase of Rice.

[0148] An alternative procedure for identification of inhibitors thataffect shikimate dehydrogenase ofX. oryzae pathovar oryzae but notshikimate dehydrogenase of rice can be followed when the sequence ofrice shikimate dehydrogenase becomes available after completion of therice genome sequence in the next year or two. The deduced amino acidsequences of the two shikimate dehydrogenase can be used to model thestructures of these enzymes using computer assisted procedures (35).Based on these structures, molecular modeling methods which are familiarto those well versed in the art of drug discovery, can then be used toidentify inhibitors/lead molecules that might inhibit shikimatedehydrogenase of X. oryzae pathovar oryzae and not affect shikimatedehydrogenase of rice.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0149]FIG. 1 shows structure of Xanthomonadin I and absorption spectraof crude pigment extracts from X. oryzae pv. oryzae cultures. (A)Structure of isobutyl derivative of xanthomonadin isolated from X.juglandis, a walnut pathogen. (B). Absorption spectra of crude pigmentextracts from BXO43 1 (wild-type strain), BXO65 (an EMS-induced pig⁻Aro⁻ Vir⁻ mutant), BXO1706 an aroE1::Tn3-HoHo1 marker exchange mutant),BXO1704 (BX065 with a complementing plasmid pAG4). The dashed verticallines indicate the wavelengths corresponding to the characteristic peakand shoulders in the absorption spectrum of xanthomanadin.

[0150]FIG. 2 shows Virulence of X. oryzae pv. Oryzae strains for rice.Inoculations are performed with greenhouse-grown plants of susceptiblerice cultivar Taichung native-1. Lesion lengths are measured 10 and 15days after inoculation. The values are means and standard deviationsbased on three independent experiments. BXO43, wild-type strain; BXO65,EMS-induced Pig⁻ Aro⁻ Vir⁻ mutant; BXO1704, BXO65 with complementingplasmid pAG4. Similar results are obtained following inoculation ofgrowth chamber-grown rice plants.

[0151]FIG. 3 shows nucleotide sequence of X. oryzae pv. Oryzae aroE geneand deduced amino acid sequence. The solid and shaded triangles indicatemTn7 and Tn3-HOHO1 insertions, respectively. The aroE3::mTn7 insertioncould not be marker exchanged. The predicted ribosome binding site (S/D)and −35 and −10 promoter regions are indicated. Restriction sites forEcoRI and HindIII are also indicated.

1. A mutant bacteria Xanthomonas orzae pv. orzae (BXO65).
 2. A mutant asclaimed in claim 1, wherein said mutant shows mutation in aroE genecoding for shikimate dehydrogenase.
 3. A mutant as claimed in claim 1,wherein said mutant is avirulent.
 4. A mutant as clamed in claim 1,wherein doubling time of said mutant is about 25.7 hours.
 5. A mutant asclaimed in claim 1, wherein said mutant produces very littleXanthomonadin pigments as compared to wild type.
 6. A mutant as claimedin claim 1, wherein said mutant does not produce aromatic amino acids.7. A method of producing a mutant bacterial strain Xanthomonas orzae pv.orzae (BXO65) of claim 1, said method comprising steps of: (a) mutatingBXO1 to produce BXO62, and (b) developing rifampin resistance in saidBXO62 to produce BXO65.
 8. A method as claimed in claim 7, wherein BXO1is wild type bacterial strain of Xanthomonas orzae pv. Orzae.
 9. Amethod as claimed in claim 7, wherein BXO62 is pigmnent deficientbacterial strain of Xanthomonas orzae pv. Orzae.
 10. A method as claimedin claim 7, wherein said bacterial strains are grown at 28° C.
 11. Amethod as claimed in claim 7, wherein said bacterial strains are grownin medium selected from a group comprising peptone sucrose (PS) medium,and modified Miller's minimal medium M4.
 12. A method as claimed inclaim 7, wherein mutation is developed by using ethyl methanesulfonate.13. A method as claimed in claim 7, wherein rifampin is used atconcentration ranging between 25 to 100 μg/ml.
 14. A method as claimedin claim 7, wherein BXO62 strain is obtained at a frequency of 1% of thesurviving mutated cells.
 15. A method as claimed in claim 7, whereinsaid method helps develop inhibitors against plant pathogenic bacteriaresponsible for Bacterial Leaf Blight (BLB) in rice.
 16. A method forscreening test compounds to identify Shikimate dehydrogenase pathwayinhibitors in bacteria to develop bactericide, said method comprising:(a) mixing shikimic acid and NADP as substrates, (b) adding testcompounds to the said mixture, (c) measuring change in absorbance at 340nm, and (d) identifying said inhibitors from the test compounds showingdecrease in absorbance.
 17. A method as claimed in claim 16, whereinabsorbance is measured spectrophotometrically.
 18. A method as claimedin claim 15, wherein mixing shikimic acid and NADP shows increase inabsorbance.
 19. A method as claimed in claim 15, wherein test compoundswith inhibitory action shows decrease in absorbance.
 20. A method asclaimed in claim 15, wherein said method is used to develop inhibitorsagainst pathogenic bacteria having shikimate dehydrogenase pathway. 21.A method as claimed in claim 15, wherein said method is used to developinhibitors against plant pathogenic bacteria responsible for BacterialLeaf Blight (BLB) in rice.
 22. A method as claimed in claim 15, whereintest compounds are selected from a group comprising extracts, compounds,biomolecules, and pharmaceutical products.